Amino acid based compositions for the treatment of pathological conditions distinguished by insufficient mitochondrial function

ABSTRACT

The present invention relates to compositions suitable for the treatment of pathological conditions distinguished by insufficient or reduced mitochondrial function. The compositions comprise, as principal active ingredients, the amino acids leucine, isoleucine and valine. The compositions may also comprise, as further active ingredients, amino acids threonine and lysine, and optionally, histidine, phenylalanine, methionine, tryptophan, as well as tyrosine and cysteine.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/575,062, filed Apr. 7, 2006, and published as US-2007/0010437 on Jan.11, 2007, which is a 371 filing of PCT International Application No.PCT/IB2004/003210, filed Sep. 30, 2004, and published in English as WO2005/034932 on Apr. 21, 2005, which claims the priority of ItalianApplication No. TO2003A000789, filed on Oct. 7, 2003, the disclosures ofwhich are hereby incorporated by reference in their entirety.

The present invention relates to compositions for the treatment ofpathological conditions distinguished by insufficient mitochondrialfunction.

Identification of specific pathologies of mitochondria date back someforty years, when a specific mitochondrial DNA was identified. However,it was only during the 1990's that a better knowledge of the synthesis,structure, and function of mitochondria was linked to specific clinicalfindings, and also to degenerative diseases correlated to age. Thanks tosaid knowledge there has been identified a series of primitive andsecondary mitochondrial diseases, as well as the various target organsof mitochondrial lesions, such as the central nervous system (and henceParkinson's disease, amyotrophic lateral sclerosis, Alzheimer'sdisease), the cardiovascular system in relation to ageing, skeletalmuscles, micro-macroangiopathy, etcetera.

The therapeutic approaches deriving from said knowledge are stillprimitive and focus, above all, on the prevention of oxidative damage(Lufth R. and London B. R.—J. Intern. Med., 1995: 238, 405-421). Infact, the production of reactive oxygen species (ROS), which are thewaste products of the oxidative metabolism, has been correlated to theprogressive accumulation of damage to DNA and proteins at various levelsin organs and cells, according to the so-called Harman hypothesis(Harman C. V.—Am. Geriatr. Soc., 1972: 20, 145-147), as well as toageing. So far the therapeutic approaches have focused on anti-oxidantsubstances, such as N-acetyl-cysteine (Martins Bañaclocha M., BrainRes., 2000: 859, 173-175), glutathione (Viria et al., 1992 in: FreeRadicals and Agents, 136-144, Birkhanserverlag, Basel), vitamin C (GhoshM. K. et al., Free Radical Res., 1996: 25, 173-179), and carnitine(Hagen T. Metal, 1998, Proc. Math. Acad. Sci. USA, 95: 9562-9666). Someexperimental results appear to confirm that the mean and maximum life ofcell lines is favourably prolonged when the amounts of anti-oxidants aresufficient to eliminate the mitochondrial oxidative stress, restoringthe metabolic activity of the mitochondria.

The present invention indicates the possibility of an absolutelyinnovative therapeutic approach to the aforesaid problem, based upon theuse of specific amounts of given amino acids.

Three quarters of the total requirements of nitrogen are covered by justfive amino acids: leucine, isoleucine, valine, threonine and lysine.This means that all the other amino acids, whether essential ornon-essential, contained in dietary proteins, serve to cover only theremaining 25% of the nitrogen requirement of the organisms of mammals.In nature there do not exist proteins with a content of amino acidsstoichiometrically similar to what is necessary for covering therequirement of nitrogen in human beings. This explains why an excessiveintake of protein in food is a possible source of many medical problems:the intake of an excess of amino acids with respect to the onesnecessary and usable will result in a functional overload of themechanisms of elimination of waste and not in an improvement of thefunctions depending upon the availability of amino acids.

In the above perspective, amino acids can be compared to letters, andproteins to words. The syntheses are activated and proceed only inconditions where there is present an adequate quantity of all thenecessary amino acids for the constitution of the entire final protein;otherwise, synthesis will not even start. Consequently, not only willthe amino acids be necessary in the right qualities, but they will alsobe necessary in adequate quantities of each, like the letters forwriting a given word. Take, for example, the Italian word “proteine”: towrite it we need one p, one r, one o, one t, one i, one n, but two e's.The correct ratio between amino acids is not expressible in grams, butin terms of number of molecules, in the correct ratio between them, andin function of the concentration of amino acids present in the proteinto be synthesized. As a result, the ratios are more correctly expressedas numbers of molecules, i.e., in gram-moles. This is the most preciseway to express the ratio, said ratio being referred to properly asstoichiometric, between amino acids necessary for the synthesis ofproteins.

A further level of complexity is due to the fact that synthesis of theproteins is an extremely expensive process from the energy standpoint;consequently, syntheses do not proceed unless there is an adequateavailability of energy in the cell, and the energy is produced by themitochondria. The production of energy is generally taken to depend uponanaerobic glycolysis of glucose and upon β-oxidation of free fatty acids(FFAs), which provide the two main substrates for maintenance ofactivity of the citric-acid cycle (aerobic glycolysis), a set of enzymesconstituting the chief producer of the energy that enables us to live,which is oxygen-dependent. Deriving from anaerobic glycolysis ispyruvate, and from this, by carboxylation, oxaloacetate, or, bydecarboxylation, acetylCoA, which, by condensing with one another, giverise to the citrate necessary for maintenance of the activity of thecycle. AcetylCoA can derive also from β-oxidation, or from themetabolism of some amino acids, which are consequently called<<ketogenetic amino acids>>, whilst pyruvate, citrate and oxaloacetatecan also be derived from the metabolism of other amino acids, referredto as <<glucogenetic amino acids>> because the liver can synthesiseglucose from these molecules. The use of substrates derived from aminoacids or FFAs enables a saving in the consumption of glucose. Glycaemia,outside of the period in which the absorption of food enables anincrease in the glucose of exogenous origin in the blood, is maintainedby the liver, with the release of glucose from the reserve deposit,i.e., glycogen, and with the continuous neo-synthesis of glucosestarting from specific amino acids (neoglucogenesis). Glycogenolysis andneoglucogenesis are always simultaneously active in the post-absorptionperiod, albeit in different ratios (just as is glycogenogenesis, in partfed by neoglucogenesis). Cells, tissues and organs, as well as the bodyitself, are open systems, in which it is necessary, and hence possible,to identify an input of information, material and energy.

On the basis of these general biochemical premises, the inventor hashypothesized that the availability of specific amino acids is able totransmit to the cells a dual message: on the one hand, the availabilityof plastic material, on the other, the availability of energeticmaterial. The inventor has moreover hypothesized that a specific degreeof availability, if adequately coupled to the duration and constancy ofthe variations of concentration, constitutes a mediator to cells of theinformation that there exists an adequate availability of material andenergy for activating the renewal or development of intracellularstructures. Consequently, for cells in which this is possible, it isuseful, or necessary, to activate cell duplication itself.

There do not exist similar hypotheses on the properties of control ofthe cell response that an adequate input is potentially able to provide.

The consequences of this approach are interesting: there exists thepossibility of identifying an energetic-metabolic lowest commondenominator that is able to maximize the response of cells, identifyingkinds and quantities of a mixture of amino acids capable of supplyingthe right message to given tissues and organs, or to the entire body.The levels of response will presumably be different within the cells,and hence within the tissues, the organs, and the body as a whole. Thecorollary to this hypothesis is the question: what may be, within thecells, the structures capable of receiving messages from theenvironment, and of transmitting to the cell, and from the cell to thetissue, and so on with increasingly greater complexity, the message thatit is possible to set under way new syntheses, or to employ sufficient,but huge, energy and plastic resources in new cells.

The attention of the inventor has thus been centred on the key organellein the production of energy, namely, the mitochondrion. A series ofrecent studies has shown that the survival of the cells and the veryduration of the life of cells, depends upon the mitochondrion. Thepresence of oxidants (ROS) produced by metabolism shortens the life ofthe mitochondrion and of the cells. A recent study has moreover shownthat, in dilative myocardiopathy, the mitochondria that are closest tothe outside of the cells are less sensitive to the oxidative insultstowards the surface (and that hence the syntheses proceed in aneccentric way), beneath the sarcolemma, than those towards the centre ofthe cells, where the mitochondria are more readily liable to undergoapoptosis (Fannin S. W. et al., Arch. Biochem. Biophys., 1999: 372,399-407). Apoptosis itself, via caspase cascade, can be triggered by themitochondria (Dirks A. and Leeuwenburg C., Am. J. Physiol. RegulatoryIntegrative Comp. Physiol., 2002: 282, R519-527).

As has been said, three quarters of the nitrogen requirement of theorganism are covered by five essential amino acids. Four of these areneutral amino acids, of which three are branched chain amino acids:leucine (ketogenetic), isoleucine (ketogenetic and glucogenetic), valine(glycogenic), and threonine (glycogenic), characterized by a hydroxylgroup in position β; the fifth is a basic amino acid, lysine, which hasan amine group (NH₃ ⁺) in position ε.

Starting from the premises set forth above, the studies underlying thepresent invention have enabled identification of a stoichiometricmixture of amino acids that would enable maximum use for syntheticpurposes, optimizing, at the same time, coverage of the energyrequirement of the mitochondrial energy metabolism. According to theinvention:

1) the mixture envisages the use of the branched amino acid leucine incombination with at least one between, and preferably both of, thebranched amino acids isoleucine and valine. The ratios, expressed ingram-moles between the amino acids, in proportion to 1 gram-mole ofL-Leucine, can be identified as follows:

L-Isoleucine: from 0.2 to 0.7, preferably from 0.4 to 0.6;

L-Valine: from 0.2 to 0.7, preferably from 0.4 to 0.6.

2) The mixture envisages, as further active ingredients, at least onebetween, and possibly both of, the amino acids threonine and lysine. Theratios, expressed in gram-moles between the amino acids, in proportionto 1 gram-mole of L-Leucine, can be identified as follows:

L-Threonine: from 0.15 to 0.50, preferably from 0.2 to 0.45;

L-Lysine: from 0.15 to 0.60, preferably from 0.3 to 0.55.

At the current state of the studies conducted by the inventor, theformulation that appears to present a greater degree of activity is aformulation in which, setting at 1 the sum of leucine, isoleucine andvaline, in the reciprocal dimensions based upon the gram molecularweight identified in point 1), the sum of threonine and lysine isbetween 10% and 50% of said formulation (always on the basis of the grammolecular weight of the substances in question), and preferably between25% and 45%.

3) The nutritional intake of the mixture can be integrated with one ormore further essential amino acids, and in particular histidine,methionine, phenylalanine, tryptophan. Setting at 1 the sum of leucine,isoleucine, valine, threonine and lysine, the other essential aminoacids (histidine, methionine, phenylalanine, tryptophan) are representedin a global amount (again expressed as gram molecular weight/grammolecular weight ratios) ranging from 2% to 25%, and preferably from 5%to 15%.

4) Two non-essential amino acids can possibly optimize the mixture ofthe aforesaid amino acids by addition thereto:

4.1) tyrosine (which is physiologically produced by hydroxylation ofphenylalanine), since derivation of tyrosine from phenylalanine canoccur only in the liver, whilst an important use occurs peripherally,for example, in muscle or in the myocardium, where the enzymatic routefor hydroxylation of said amino acid does not exist; the optimal amountof tyrosine has been identified as one ranging from 15% to 50% of theamount of phenylalanine present in the mother mixture, and preferablyfrom 20% to 35%. The greater the hepatic impairment, or the peripheralrequirement as a function of a reduced intake from other sources, thegreater will be the usefulness of increasing the stoichiometric ratiosbetween tyrosine and phenylalanine, up to an indicative maximum of 50%;

4.2) cyst(e)ine (cystine and/or cysteine), which will preferably be atleast 100%, with an optimal amount identified as being comprised between150% and 350%, of the amount of methionine present in the mothermixture.

The aforesaid presence of cysteine, which can be readily transformedmetabolically into methionine, will prevent the relative shortage ofcysteine, in the process of interconversion of methionine into cysteine,from giving rise, in conditions of relative excess of methionine orshortages of folates, to the toxic intermediate, i.e., homocysteine, andstopping at that point.

5) One or more other amino acids may be envisaged as further activeingredients of the mixture, the sum in gram molecular weight of whichwill be in a percentage lower than 20% with respect to the other activeingredients, and less than 10% for each individual additional aminoacid.

6) In its preferred formulation, the mixture according to the inventionhas a pH in aqueous solution of between 6.5 and 8.5, whether with orwithout excipients suitable for the preparation of tablets, capsules,powders, etc., and in any pharmacological form of presentation suitablefor enteral or parenteral use.

Further specifications, in terms of amounts and ratios between thevarious amino acids envisaged by the compositions according to theinvention, are contained in the attached claims, which constitute anintegral part of the present description. Even though the ratios areexpressed on the basis of molecular weight, the ones indicated in theattached claims are applicable, in general terms, also in the case ofcalculation based on the weight in grams of the various amino acidsindicated (bearing, however, in mind that the amount of lysine,expressed in grams rather than in moles, may then be greater than theindividual amounts of isoleucine and valine).

An example of formulation of the mixture of amino acids according to theinvention, made in accordance with the principles indicated, is given inthe following table:

TABLE 1 Molec. Amino acid Weight* g/100 g % of total % of clusterL-Leucine 131.17 31.2500  31.25%  50.00% L-Isoleucine 131.17 15.6250 15.63%  25.00% L-Valine 117.15 15.6250  15.63%  25.00% Totals-Cluster A62.5000  62.50% 100.00% L-Lysine 146.19 16.2500  16.25%  65.00%L-Threonine 119.12 8.7500  8.75%  35.00% Totals-Cluster B 25.0000 25.00% 100.00% L-Histidine 155.16 3.7500  3.75%  46.88% L-Phenylalanine165.19 2.5000  2.50%  31.25% L-Methionine 149.21 1.2500  1.25%  15.63%L-Tryptophan 204.23 0.5000  0.50%  6.25% Totals-Cluster C 8.0000  8.00%100.00% L-Tyrosine 181.19 0.7500  0.75% L-Cystine 240.30 3.7500  3.75%Totals for composition 100.0000 100.00% *from <<Amino Acids, NucleicAcids & Related Compounds-Specification/General Tests>>, 8^(th) Edition,Kyowa Hakko Kogyo Co., Ltd.

In the following table, the amount in grams of the composition referredto in Table 1 are expressed on the basis of molecular weight.

TABLE 2 Molec. Amino acid Weight moles % of total % of cluster L-Leucine131.17 0.23824  31.97%  48.55% L-Isoleucine 131.17 0.11912  15.98% 24.27% L-Valine 117.15 0.13338  17.90%  27.18% Totals-Cluster A 0.49074 65.85% 100.00% L-Lysine 146.19 0.11116  14.92%  60.21% L-Threonine119.12 0.07346  9.86%  39.79% Totals-Cluster B 0.18461  24.77% 100.00%L-Histidine 155.16 0.02417  3.24%  48.21% L-Phenylalanine 165.19 0.01513 2.03%  30.19% L-Methionine 149.21 0.00838  1.12%  16.71% L-Tryptophan204.23 0.00245  0.33%  4.88% Totals-Cluster C 0.05013  6.73% 100.00%L-Tyrosine 181.19 0.00414  0.56% L-Cystine 240.30 0.01561  2.09% Totalsfor composition 0.74522 100.00%

As will emerge clearly hereinafter, the administration of a mixtureaccording to the invention, and in particular according to Table 1, isdecisive in increasing the number of mitochondria. On the one hand, thisleads to an inhibition of apoptosis, via a precise caspase-mediatedmechanism, which is the inhibition of activation of the caspasespecifically resulting from mitochondrial suffering; on the other hand,it leads to an increase in the availability of energy for eachindividual cell, and hence to the clinical improvement referred to.

The mixture is functional for achieving this result by virtue ofpeculiar stoichiometric ratios between “clusters” of amino acids, whichcan be inferred also from Table 2. It should be noted that these are notnecessarily chemico-physical “clusters”, nor do they have any othercommon metabolic characteristics except for the final result at whichthe peculiar stoichiometric ratios aim: covering the energyrequirements, and in any case allowing availability of amino-acidsubstrate for the necessary processes of synthesis. It is thesimultaneous availability for satisfying both of these needs whichactuates the anti-apoptosis and synthetic command.

CLINICAL STUDIES

The problem was approached in two different ways, i.e., from theclinical standpoint and from the experimental standpoint, proceeding ina logical sequence.

In the knowledge that in the elderly there occurs a reduction in thenumber of mitochondria in the muscular structure or mass, a clinicalstudy was conducted to verify whether the beneficial effects ofsupplementing with the mixture according to Table 1 could be confirmedin elderly subjects with problems of limitation of mobility. In thistype of patients, a reduced mobility creates disability in movement,causing loss of muscular trophism, and this in turn, in a viciouscircle, brings about a further reduction of mobility. In these patients,the incapacity to adapt their cardiac function to the greater workloadrequired by physical activity can become one of the limiting factors.

There were consequently enrolled 40 patients aged over 65 years, who leda sedentary life and suffered from a low quality of life in relation totheir state of health, with a normal ventricular ejection (VE) at rest,without angina, or any other invalidating pathological condition. Theirreduced physical activity was documented by a 6-minute walking test, andtheir perception of difficulty of deambulation via a questionnaire oninvalidity in walking. In addition, using a dynamometer, the maximumisometric muscular force was measured. VE was moreover measured at restand during isometric exercise with two-dimensional echocardiography. Thedata were evaluated prior to and at the end of 3 months of oraladministration of the mixture of amino acids according to Table 1.

Body Mass Index (BMI) was not modified by the therapy, but the distancecovered in a 6-minute walk increased from 214.5±32 metres to 262.8±34.8metres (P<0.001) after the treatment, and there was likewise animprovement in the data of the self-assessment questionnaire, as regardsdistance, speed and number of flights of stairs (P<0.001, with respectto the basal values). VE at rest (normal: >50%) was not modified by thetherapy, nor was blood pressure or heart rate.

The capacity for isometric exercise measured with a dynamometer(handgrip test), instead, increased from 16.6±2.4 to 19.2±2.2 (P<0.001).Under stress, VE should not vary or increase in normal conditions, bymore than 0.05 U. Prior to start of therapy, 66% of the patients showeda decline in VE under stress (P>0.005), whilst the remaining 34% had anormal response. In 93% (P<0.001) of the patients with altered VE, afterthree months of therapy, VE under stress increased or remained unchangedwith respect to the basal value, i.e., it had normalized.

Various experimental models were adopted to interpret and explain themechanism through which the aforesaid mixture of amino acids were ableto activate a better energetic performance of the muscular cells of themyocardium in the clinical study described.

A first experimental model, using the perfused isolated heart of a rat,furnished indirect indications, which enabled confirmation of thehypothesis that the mitochondrion was the organelle to be studied aspossible actor in the attainment of the results found. For this purpose,in the isolated heart of a rat, perfused with a standard buffersolution, a perfusion phase of 30 minutes was followed by a phase ofcomplete ischaemia (ligation of the coronaries) and asystolia protractedfor 30 minutes; then, the myocardium was re-perfused and made to startbeating again.

The groups were three:

a) a control group;

b) a group similar to the controls, but with an acute perfusion with themixture referred to in Table 1, in an amount of 0.25 mg/mL in the phaseof 30 minutes that preceded ischaemia and in that of post-ischaemicre-perfusion;

c) a group of animals treated for 20 days with 1 g/kg/d of the mixturereferred to in Table 1, prior to isolation of the myocardium.

The variables measured were:

-   -   systolic and diastolic arterial pressure (PA);    -   release of creatinphosphokinase (CPK) and lactate (enzymatic        tests);    -   mitochondrial activity (with Clark's electrode);    -   apoptosis of endothelial cells and of cardiomyocytes (with        TUNEL);    -   the activity of caspase-3 and caspase-9 in the endothelium and        in the cardiomyocytes (with the fluorimetric test).

The results can thus be summed up as described in what follows.

Group <<a>> (controls) and group <<b>> (acute perfusion in an untreatedanimal) showed results altogether similar for all the parametersexamined, and hence no effect. Instead, the hearts of animals of group<<c>> (pre-treated for 20 days with 1 g/kg/d of the mixture referred toin Table 1 prior to isolation of the myocardium) presented a clearlylower diastolic arterial pressure (PA), as well as a functional responseto re-perfusion and a clearly higher recovery of ventricularcontraction.

Likewise, there was a significant reduction in the release of CPK, whichindicates a greater integrity of the cell membranes, and in the releaseof lactate, said reduction indicating a greater use of pyruvate by thecitric-acid cycle, albeit in total absence of oxygen, since it was nolonger arriving with the blood.

At this point, a check was made to see whether there was any increase inthe production of energy, and how this could influence myocardialcontractility during a protracted ischaemic phase, and in there-perfusion phase.

Using the same methodology, and the same treatment groups, there wasthen studied the content of ATP and creatine phosphate (CF) in thetissue, as well as mitochondrial activity, evaluating the VO₂ and theproduction of mitochondrial ATP.

Only the chronic treatment (group <<c>>: 1 g/kg/d of the mixturereferred to in Table 1 prior to isolation of the myocardium) gave riseto statistically significant modifications of the parameters examined,inducing an improvement, i.e., increase, in the activity of themitochondria.

Since the integrity of the mitochondria is fundamental for themaintenance of the integrity of living cells, it was of particularinterest to identify whether the mixture referred to in Table 1 had aneffect on apoptosis, and in order to understand the possible mechanismsof control different levels of cascade of caspases, i.e., the enzymaticmechanisms controlling intracellular apoptosis, were studied.

Since the mitochondrion is the organelle that produces energy, it isfundamental for the life of the cell. The loss of mitochondria willresult in a progressive loss of the energy assets of the cell, withprogressive impairment of the processes of elimination of wastematerial, of synthesis, and of cell function. The cell becomes less andless able to maintain itself; consequently, the process of apoptosis (aform of cell suicide) takes place to reduce the number of inefficientcells and to give to the others the possibility of replacing them or ofsurviving in conditions of lower competition on the substrates.Apoptosis is controlled by various mechanisms, some of which areextracellular, for activation of specific receptors, and some of whichare intracellular, of which the principal one—which is perhapsindispensable post-receptor mediator also for the others—is undermitochondrial control. Apoptosis of mitochondria releases cytochrome cinto the cytoplasm, and this activates caspase-9, which is followed bydirect activation of the remaining apoptotic cascade.

This notation is of particular importance for an understanding of themechanism of action of different therapeutic approaches on apoptosis:the control of the mitochondrion on caspase-9 is specific and, hence, inorder to be certain not to have created random events or possibleinterference with other mechanisms of activation of different caspases,also caspase-8 was studied (activation of which is principally linked toIL-1 and to TNFα).

Caspase-9 (dosed in a blind way by an external laboratory) was the onlyone to show a significant reduction in its presence in the animals ofgroup <<c>>, which were treated chronically via oral route for 20 days,with 1 g/kg/d of the mixture referred to in Table 1 prior to isolationof the myocardium. No modification was observed in the study ofcaspase-8, activated by membrane receptors.

This series of studies makes it possible to understand how the resultsobserved experimentally and in the clinical study in humans referred topreviously is necessarily to be ascribed to the mitochondrion,independently of receptor activation and extracellular influences.

Finally, then, it was verified whether the greater cellular metabolicefficiency was due to an increased activity of individual mitochondriaor else—and this is an event known to be indispensable during cellduplication, being historically well documented and filmed inexperimental observations (Padoa E. 1967, in: Biologia Generale, 3rdedition. Chapter 4: La cellula, 116-189, Boringhieri Editore S.p.A.,Turin), but never even hypothesized as being inducible in physiologicalor pathological conditions—whether the mitochondria were multiplied,increasing their number, in response to the introduction of the mixtureof amino acids according to the invention.

This led to the need to count the number of mitochondria, before andafter treatment, in pathological conditions that reduced the numberthereof, and similar to those in which the study in humans had beenconducted.

Eighteen-month old Wistar rats were taken, checked at 22 and 24 months.A control group was compared to a group that was treated with themixture of amino acids according to the invention, adopting arandomization criterion. First of all, the number of mitochondria (MT)was counted independently, using histochemical and ultrastructuralmethods (by electronic microscopy) in six-month old animals, ascontrols. The number of the mitochondria in the peripheral muscle, inthe myocardium, and in the cells of the cerebral cortex, wasproportionally reduced according to age (i.e., a smaller number in theolder animals).

Administration of the mixture referred to in Table 1 of 0.3 g/kg/d⁻¹ tothe senescent animals proved extremely efficient in maintaining thepatrimony of mitochondria more intact, i.e., higher, by a percentagevalue of 26±5% and 31±7%, respectively, than for the untreated animalsof comparable age in the checks carried out at 22 months and at 24months.

From this it is shown that increasing the number of mitochondria is notonly possible but certainly therapeutic in various conditions.

Subsequent analyses enabled ascertainment of the fact that animprovement of 23±4% in the number of mitochondria was possible withjust five amino acids of the mixture of Table 1, using a stoichiometricratio of approximately 1:0.5 between the sum of leucine, isoleucine andvaline (1), and the sum of threonine and lysine (0.5). With the removalof threonine and/or lysine, the number of mitochondria was not modifiedsignificantly in any of the animals. The reduction of leucine to below15% of the total weight of the mixture led to nullifying significantlythe positive effects of the mixture, whilst the effects were lessmarked, albeit statistically significant, if just isoleucine or justvaline were reduced in the same proportion from the mixture, providedthat the leucine was maintained constant (in said perspective, thecompositions according to the invention may envisage up to 60% ofleucine but not less than 15% with respect to the other two branchedamino acids, up to 40% of isoleucine, but not less than 15% with respectto the other two branched amino acids, and up to 40% of valine, but notless than 15% with respect to the other two branched amino acids).

The preliminary studies carried out, in which different types ofconditions regarding nutritional intake, and to the amount of physicalexercise imposed, enabled confirmation of how, in volunteers, thestoichiometric ratios as claimed between the different amino acids takeinto account the most extreme needs, minimizing the possibility of anindividual amino acid being found in excess, or deficient, in thereciprocal ratio with the others, and that said ratios enable maximumuse of the amino acids for, synthetic purposes, optimizing, at the sametime, the coverage of the energy requirements of the mitochondrialenergy metabolism.

From the foregoing, it emerges clearly how the compositions according tothe invention prove useful for the treatment of pathological conditionsdistinguished by insufficient mitochondrial function in humans and inanimals, such as sarcopenia of the aged and senescence, in so far asthey are designed to maintain intact and/or restore and/or increase thenumber of intracellular mitochondria, as well as to modify in a positivesense the activity of production of intracellular energy. Thecompositions according to the invention thus prove useful in everycondition in which a reintegration of a reduced cellular energeticactivity may modify to advantage the activity of the cell itself, andhence of the tissue and organ that is formed by the set of cells, suchas an increase of neuronal activity in degenerative diseases of thenervous system (Alzheimer's disease, amyotrophic lateral sclerosis,Parkinson's disease), in which the activity of the cells decreases onaccount of reduced energy capacity, which involves a loss of themitochondrial energetic activity. Likewise, the compositions accordingto the invention are suitable for use in every condition in whichischaemic states of any aetiology inhibit the energetic activity of thecell, promoting the maintenance of the structures designed to produceenergy, and in particular the integrity of the mitochondria. Thecompositions also prove advantageous in every condition in which it isuseful to the function of the organ to antagonize the apoptoticphenomena of the mitochondria and controlled by the mitochondria, whichare not linked to apoptosis mediated by cell-membrane receptors and bymeans of other caspases that are not the ones activated by caspase-9.

The field of application of the invention extends to proteins obtainedfrom genetic engineering or any other artificial method, in which thereis a stoichiometric composition of amino acids as described above andclaimed in the annexed claims.

In order to implement the invention, the amino acids indicated above canbe replaced by respective pharmaceutically acceptable derivatives,namely salts.

1. A method for treating Alzheimer's disease in a subject, said methodcomprising: administering via oral route to the subject atherapeutically effective amount of a composition comprising, as activeingredients, the following: (i) the branched chain amino acids leucine,isoleucine, and valine, and/or salts thereof; (ii) lysine and threonine,or salts thereof; and (iii) other essential amino acids selected from:histidine, methionine, phenylalanine, and tryptophan, or salts thereof,wherein the amount in moles of threonine is smaller than the individualamounts of lysine and of said branched amino acids, or salts thereof,but greater than the sum of the individual amounts in moles of saidother essential amino acids, or salts thereof; and the amount in molesof lysine is smaller than the individual amounts of said branched aminoacids, or salts thereof, but greater than the sum of the individualamounts in moles of said other essential amino acids, or salts thereof.2. The method according to claim 1, wherein isoleucine, valine,threonine, and lysine are present in the following molar ratios toleucine: isoleucine/leucine having a molar ratio from 0.2 to 0.7;valine/leucine having a molar ratio from 0.2 to 0.7; threonine/leucinehaving a molar ratio from 0.15 to 0.50; and lysine/leucine having amolar ratio from 0.15 to 0.60.
 3. The method according to claim 1,wherein the sum of the amounts in moles of histidine, methionine,phenylalanine, tryptophan, or salts thereof, is from 2% to 25% of thesum of the amount in moles of leucine, isoleucine, valine, lysine, andthreonine, or salts thereof.
 4. The method according to claim 1, whereinthe composition further comprises, as an active ingredient, at least oneof tyrosine and cyst(e)ine, or salts thereof.
 5. The method according toclaim 1, wherein the composition further comprises tyrosine or a saltthereof, and wherein the amount in moles of tyrosine or salt thereof isfrom 15% to 50% of the amount in moles of phenylalanine or salt thereof.6. The method according to claim 1, wherein the composition furthercomprises cyst(e)ine or a salt thereof, and wherein the amount in molesof cyst(e)ine or salt thereof is at least equal to 100% of the amount inmoles of methionine or salt thereof.
 7. The method according to claim 4,wherein the composition further comprises both tyrosine and cyst(e)ine.8. The method according to claim 3, wherein the sum of the individualamounts in moles of threonine and lysine, or salts thereof, is smallerthan the sum of the individual amounts in moles of said branched aminoacids, or salts thereof, but greater than the sum of the individualamounts in moles of the said other essential amino acids, or saltsthereof.
 9. The method according to claim 3, wherein the amount in molesof threonine, or salt thereof, is smaller than the individual amounts inmoles of lysine and of said branched amino acids, or salts thereof, butgreater than the individual amounts in moles of said other essentialamino acids, or salts thereof.
 10. The method according to claim 3,wherein the amount in moles of lysine, or salt thereof, is smaller thanindividual amounts in moles of said branched amino acids, or saltsthereof, but greater than the individual amounts in moles of said otheressential amino acids, or salts thereof.